Bottom Line:
However, the potential adverse effects of surface-functionalized CNTs have not been well characterized.The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB.These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways.

ABSTRACTBiomedical applications of carbon nanotubes (CNTs) often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs) via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol) linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS) involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47(phox) and p67(phox) in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity.

pone-0065756-g007: MWCNTs-COOH and MWCNTs-PEG induced ROS generation in RAW 264.7 cells.Cells were treated with or without 75 µg/mL of MWCNT samples for the indicated times, and the ROS levels were measured by (A) DCF staining and (B) HE staining using a flow cytometer or (C) fluorescence microscope. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. Hydrogen peroxide (H2O2) (1 mM) was used as the positive control. *p<0.05 compared to control sample, #p<0.05 compared to MWCNTs-PEG. Data represent similar results from three independent experiments.

Mentions:
To elucidate whether intracellular accumulation of ROS contributes to apoptosis of macrophages induced by MWCNTs-COOH and MWCNTs-PEG, the fluorophore H2DCF-DA was used to detect ROS generation following exposure of RAW 264.7 cells to 75 µg/mL of either MWCNTs-COOH or MWCNTs-PEG for up to 24 h. As shown in Figure 7A and C, MWCNTs-PEG showed less induction of ROS generation as compared with MWCNTs-PEG after 12 h of incubation. To specify the nature of ROS, the superoxide-specific dye DHE was used to detect superoxide generation. As observed with DCF staining, treatment with MWCNTs-PEG induced much less accumulation of superoxide radicals than MWCNTs-COOH (Figure 7B, C).

pone-0065756-g007: MWCNTs-COOH and MWCNTs-PEG induced ROS generation in RAW 264.7 cells.Cells were treated with or without 75 µg/mL of MWCNT samples for the indicated times, and the ROS levels were measured by (A) DCF staining and (B) HE staining using a flow cytometer or (C) fluorescence microscope. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. Hydrogen peroxide (H2O2) (1 mM) was used as the positive control. *p<0.05 compared to control sample, #p<0.05 compared to MWCNTs-PEG. Data represent similar results from three independent experiments.

Mentions:
To elucidate whether intracellular accumulation of ROS contributes to apoptosis of macrophages induced by MWCNTs-COOH and MWCNTs-PEG, the fluorophore H2DCF-DA was used to detect ROS generation following exposure of RAW 264.7 cells to 75 µg/mL of either MWCNTs-COOH or MWCNTs-PEG for up to 24 h. As shown in Figure 7A and C, MWCNTs-PEG showed less induction of ROS generation as compared with MWCNTs-PEG after 12 h of incubation. To specify the nature of ROS, the superoxide-specific dye DHE was used to detect superoxide generation. As observed with DCF staining, treatment with MWCNTs-PEG induced much less accumulation of superoxide radicals than MWCNTs-COOH (Figure 7B, C).

Bottom Line:
However, the potential adverse effects of surface-functionalized CNTs have not been well characterized.The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB.These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways.

ABSTRACTBiomedical applications of carbon nanotubes (CNTs) often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs) via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol) linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS) involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47(phox) and p67(phox) in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity.